© 2012 Ma ín-Bande as e al, publishe and licensee Do e Medical P ess L d. This is an Open Access
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In e na ional Jou nal o Nanomedicine 2012:7 5793–5806
In e na ional Jou nal o Nanomedicine
Cannabinoid de i a e-loaded PLGA nanoca ie s
o o al adminis a ion: o mula ion,
cha ac e iza ion, and cy o oxici y s udies
Lucía Ma ín-Bande as1
Jose a Ál a ez-Fuen es1
Ma ilde Du án-Loba o1
José P ados2
Consolación Melguizo2
Me cedes Fe nández-
A é alo1
Mª Ángeles Holgado1
1Depa men o Pha macy and
Pha maceu ical Technology, Facul y o
Pha macy, Uni e si y o Se ille, Se ille,
Spain; 2Ins i u e o Biopa hology and
Regene a i e Medicine (IBIMER),
School o Medicine, Uni e si y o
G anada, G anada, Spain
Co espondence: Mª Ángeles Holgado
Depa men o Pha macy and
Pha maceu ical Technology, Facul y
o Pha macy, Uni e si y o Se ille c/
P o eso Ga cía González n° 2,
41012 Se ille, Spain
Tel +34 954 551 624
Fax +34 954 556 085
Email [email p o ec ed]
Abs ac : CB13 (1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone)-loaded
poly(lac ic-co-glycolic acid) nanopa icles (NPs) we e p oduced by nanop ecipi a ion and es ed
o hei in i o elease beha io and in i o cy o oxici y assays. The e ec s o se e al o mula ion
pa ame e s such as polyme ype, su ac an concen a ion, and ini ial d ug amoun we e s udied.
NPs had a pa icle size 90–300 nm in diame e . Resul s ob ained show ha he main in luence
on pa icle size was he ype o polyme employed du ing he pa icle p oduc ion: he g ea e
he hyd ophobici y, he smalle he pa icle size. In e ms o encapsula ion e iciency (%), high
alues we e achie ed (∼68%–90%) o all o mula ions p epa ed due o he poo solubili y o
CB13 in he ex e nal aqueous phase. Mo eo e , an in e se ela ionship be ween elease a e and
NP size was ound. On he o he hand, low molecula weigh and low lac ide con en esul ed in a
less hyd ophobic polyme wi h inc eased a es o wa e abso p ion, hyd olysis, and e osion. NPs
showed no cy o oxici y and may be conside ed o be app op ia e o d ug-deli e y pu poses.
Keywo ds: neu opa hic pain, CB13, nanop ecipi a ion
In oduc ion
Nano echnology has become one o he mos in ensi ely s udied esea ch a eas in ecen
imes. Ma e ials a he scale o a nanome e ha e unique physicochemical p ope ies
ha a e due o hei small size, su ace a ea, chemical composi ion, su ace s uc u e,
solubili y, and shape.
O e he las se e al decades, nume ous nanoca ie pla o ms ha e been s udied
o hei use as he apeu ic agen s, wi h g ea en husiasm, in bo h academic and indus-
ial applica ions. These nanoca ie pla o ms include liposomes, polyme he apeu ic
conjuga es, polyme ic micelles, dend ime s, nanoshells, and nucleic acid-based
nanopa icles (NPs). These polyme ic nanoca ie s bea g ea po en ial o biomedi-
cal applica ions owing o hei biomolecula design and small size, and a e known
o ha e shown exci ing esul s in p eclinical s udies demons a ing hei po en ial
as he apeu ic ca ie s.1 Among he polyme ic ca ie s, poly(lac ic-co-glycolic acid)
(PLGA) has a g ea po en ial in applica ions combining a ge ing, imaging, diagnos ics,
and he apy.2 Fu he mo e, as is known, his polyme can easily be hyd olyzed in o
indi idual monome s (lac ic acid o glycolic acid), which a e hen emo ed om he
body ia no mal me abolic pa hways.3
Howe e , low wa e solubili y o hese d ugs limi s hei o al bioa ailabili y and
abso p ion. The e is hus an u gen need o adequa e op ions o deli e hese d ugs
o he pa ien . Al hough he e a e di e se s a egies (use o cosol en s, sal o ma ion,
complexes wi h cyclodex ins, e c) o sol e his p oblem,4 se e al nano echnology-based
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Do ep ess 5793
ORIGINAL RESEARCH
open access o scien i ic and medical esea ch
Open Access Full Tex A icle
h p://dx.doi.o g/10.2147/IJN.S34633
Numbe o imes his a icle has been iewed
This a icle was published in he ollowing Do e P ess jou nal:
In e na ional Jou nal o Nanomedicine
22 No embe 2012
In e na ional Jou nal o Nanomedicine 2012:7
d ug deli e y sys ems ha e eme ged o inc ease he bioa ail-
abili y o nume ous d ugs ha a e poo ly soluble in wa e .5,6
Cu en ly, he e a e a conside able numbe o nanobased
d ug-deli e y sys ems being de eloped by a ious pha ma-
ceu ical companies. A comp ehensi e e iew o s a egies o
imp o e he o al bioa ailabili y o his ype o d ugs can be
ound in Fasinu e al.7
The e ha e been con o e sial indica ions as o he ex en
and mechanism o anspo o hese nanoca ie s, bu he e
is now no dispu e o e he ac ha pa icula e up ake does
ake place, especially ia he Peye ’s pa ches M-cells and
isola ed ollicles in gu -associa ed lymphoid issue, and also
ia he no mal en e ocy es.8
In his pape , he cannabinoid 1-Naph halenyl
[4-(pen yloxy)-1-naph halenyl]me hanone (CB13), which
ac s as a po en agonis a bo h he CB1 and CB2 ecep o s,
is used as a model d ug. This compound, in beha io al animal
models ( a and guinea pig) o ch onic pain (neu opa hic
and nocicep i e), has been shown o e e se es ablished
mechanical hype algesia a e bo h o al adminis a ion and
local injec ion in o a hind-paw. In beha io al es s o cen al
ne ous sys em (CNS) ac i i y in a , CB13 p oduced signi i-
can CNS e ec s only a doses ha we e 20- old highe han
he o al doses equi ed o e e se hype algesia. Thus, hese
da a indica e ha CB13 p oduces an ihype algesic ac i i y
p edominan ly ia an ac ion on pe iphe al senso y ne es.9
Howe e , CB13 is highly lipophilic and belongs o he
class 2 compounds (low solubili y and a high pe meabili y) o
he Biopha maceu ics Classi ica ion Sys em, showing a low
wa e solubili y (∼0.001–0.002 mg/mL).9 As a consequence
o i s poo solubili y and dissolu ion in he gas oin es inal
luids, his compound is incomple ely abso bed.9 Hence, his
d ug was selec ed on he basis o i s he apeu ic in e es and
inadequa e physicochemical p ope ies. I is expec ed ha , by
including CB13 in a PLGA nanoca ie , p oblems associa ed
wi h low wa e solubili y will disappea . This way, CB13 o al
iabili y would be imp o ed.
Hyd ophobic d ugs a e usually encapsula ed in biodeg ad-
able polyme s using one o h ee me hods: homogeniza ion,
sonica ion, o nanop ecipi a ion. An ideal me hod would
p oduce NPs wi h he ollowing cha ac e is ics: na ow size
dis ibu ion; size in he ange 100–1000 nm (wi h unimodal
popula ion); capaci y o high d ug inco po a ion; con olled
d ug con en o e a wide ange; non oxic; and ela i ely
easily p oduced.10 The selec ed p epa a ion me hod o NPs
was he nanop ecipi a ion me hod, also known as sol en
displacemen me hod. I is based on in e acial deposi ion o
a polyme a e displacemen o a semipola sol en , miscible
wi h wa e , om a lipophilic solu ion. Rapid di usion o he
sol en in o he aqueous phase esul s in a dec ease in he
in e acial ension be ween he wo phases, which inc eases
he su ace a ea and leads o he o ma ion o small d ople s
o o ganic sol en , e en wi hou any mechanical s i ing.11
Mo eo e , his me hod p o ides high encapsula ion e iciency
(EE) o d ugs p esen ing low wa e solubili y.3
So, he main objec i es o he p esen s udy we e: (1) o
p oduce CB13-PLGA loaded NPs using nanop ecipi a ion
as he mos sui able echnique o syn hesis; (2) o ealize he
in i o cha ac e iza ion o he NPs; and (3) o de e mine he
in i o cy o oxici y o NPs and e alua e hei sa e y and
po en ial use as nanoca ie s o o al d ug adminis a ion.
Me hods
Ma e ials
CB13 was ob ained om Toc is Cookson L d (B is ol, UK).
PLGA 50:50 was ob ained in di e en o mula ions,
Resome ® RG 502 (molecula weigh [Mw]: 12,000; inhe -
en iscosi y: 0.24 dL/g), Resome RG 502H (Mw:12,000;
inhe en iscosi y: 0.19 dL/g), Resome RG 504 (Mw:48,000;
inhe en iscosi y: 0.5 dL/g), Resome RG 504H (Mw:
48,000; inhe en iscosi y: 0.53 dL/g) and PLGA 75:25,
and Resome RG 752S (Mw: 15.000; inhe en iscosi y:
0.24 dL/g), om Boeh inge Ingelheim Gmbh (Ingelheim,
Ge many).
The su ac an s employed, Span® 60 and Plu onic® F-68,
we e ob ained om Sigma-Ald ich (S Louis, MO). Ace one
PRS was pu chased om Pan eac Química (Ba celona,
Spain); p opylene glycol (PPG) and glyce ol we e ob ained
om Aco a ma Dis ibución SA (Ba celona, Spain).
The e e sed phase high-pe o mance liquid ch oma-
og aphy (RP-HPLC) analysis was ca ied ou on a Hi achi
LaCh om® Se ies HPLC sys em (D-7000; Hi achi L d, Tokyo,
Japan) equipped wi h an L-7200 au oma ic injec o , an
in e phase D-7000 and a qua e na y pump (model L-7100),
and DAD UV–VIS de ec o (model L-7455). A Wa e s
Co p Sphe iso b ODS2 column (10 µm, 4.6 mm × 250 mm;
Mil o d, MA), kep a 40.0°C ± 0.1°C (Eli e LaCh om L-2350
column o en; Hi achi L d), was used in his analysis. Da a
collec ion and calcula ion we e done by using HSM D-7000
LaCh om® so wa e (Hi achi, L d).
P epa a ion o CB13-loaded PLGA NPs
The NPs we e p epa ed by he nanop ecipi a ion me hod
wi h modi ica ion.11 B ie ly, a weighed amoun o PLGA
was codissol ed wi h Span 60 in ace one o each a 1.5%
w/ concen a ion. Then, 5 mL o such solu ion was added
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d opwise a di e en a es (30, 15, and 5 mL/min) using a
sy inge pump (Ha a d Appa a us L d, Edenb idge, UK)
in o 15 mL Plu onic F68 aqueous solu ion (0.5% w/ ) unde
magne ic s i ing. The ace one was hen e apo a ed a oom
empe a u e (RT) o 4 hou s.
The pa icle suspension was cen i uged o e a glyce ol
bed (100 µL) a 10,000 pm o 15 minu es a 4°C o collec
he NPs. A e washing wice, he NPs we e esuspended
in he c yop o ec an solu ion and hen ozen in liquid
ni ogen and lyophilized (C iodos-50; Tels a Indus ial SL,
Te assa, Spain), a −80.0°C ± 0.5°C, and 0.057 mba , o
ob ain a ine powde . All he o mula ions we e p epa ed
in iplica e (n = 3).
Fo CB13-PLGA loaded NPs, he polyme and d ug
we e codissol ed in ace one a di e en concen a ions
(6, 10, and 20% w/w).
Cha ac e iza ion me hods
The mean diame e and size dis ibu ion o CB13 loaded-
PLGA NPs we e measu ed a 25.0°C ± 0.5°C by a lase sca e -
ing echnique based on Mie heo y (Pa ica LA-950V2; Ho iba
L d, Kyo o, Japan). An aliquo o 1 mL o ecen ly p epa ed
pa icles was dilu ed in a 12 mL cell. Measu emen s we e ca -
ied ou unde con inuous magne ic agi a ion. The in luences
o (1) o ganic phase addi ion a e in o he aqueous phase,
(2) he ype o polyme , and (3) d ug loading we e s udied.
NP aspec and mo phology we e s udied by scanning
and ansmission elec on mic oscopy (SEM and TEM).
The shape and mo phology cha ac e is ics o he NPs we e
de e mined by SEM (XL-30; Royal Philips Elec onics,
Ams e dam, The Ne he lands) a e coa ing lyophilized
samples wi h a gold hin ilm. Fo TEM analysis (CM-10;
Philips), a sample o lyophilized pa icles was suspended in
dis illed wa e . An aliquo o his suspension was d opped
on a g id and d ied a RT.
NPs su ace cha ge was de e mined by ze a po en ial (ZP)
measu emen s. The ZP o he pa icles was de e mined by
lase Dopple (Ze amas e 300; Mal e n Ins umen s L d,
Mal e n, UK). ZP measu emen s we e ca ied ou in iplica e
a e washing he NPs wi h dis illed wa e a RT. The e ec s
o polyme ype and d ug loading we e s udied.
Ch oma og aphic condi ions
The in oduc ion o new HPLC me hods o ou ine qual-
i y con ol o pha maceu ical p epa a ions s a s wi h he
es ablishmen o he op imal measu emen condi ions and
p o ides he maximum ele an in o ma ion by analyzing
he expe imen al da a.12–14
The ch oma og aphic condi ions15,16 we e de e mined
using a column C18 (Sphe iso b® 5 µm ODS2, 4.6 ×
250 mm Analy ical Column; Wa e s Co p). The mobile phase
consis ed o wo sol en s:
• Sol en A: ace oni ile:wa e :ace ic acid (75:23.7:1.3 / )
• Sol en B: ace oni ile
These sol en s we e main ained in a low a e o 1.000 mL/min:
70%:30% (A:B). The mobile phase was il e ed h ough
a 0.22 µm ni ocellulose-memb ane il e (Me ck Mil-
lipo e, Bille ica, MA) and degassed unde acuum p io
o use. A calib a ion cu e was cons uc ed using s anda d
CB13 solu ions in ace one. The calib a ion cu e was lin-
ea be ween 2.5 and 750 µg/mL (y = 17,268 ⋅ x + 19,0397,
R2 = 0.9900, F = 583.78, P = 3.25 ⋅ 1 0 −3). The limi s o de ec-
ion and quan i ica ion we e 0.5 µg/mL and 1.25 µg/mL,
espec i ely.
The de ec ion wa eleng h UV was 230 nm and he injec-
ion olume was 10 µL. The ope a ing empe a u es we e
main ained a RT, bu he o en was hea ed a 40°C o a o
he mobile phase low h ough he column.
De e mina ion o Plu onic F68 esiduals
The mos popula s abilize o he p oduc ion o PLGA-
based NPs is poly( inyl alcohol), which is di icul o emo e
om he NP su ace and has been epo ed o be po en ially
oxic.17,18 In he p esen wo k, Plu onic F68 was employed
o s abilize he NPs and could be conside ed as a aluable
al e na i e o poly( inyl alcohol) and a p omising FDA-
app o ed su ace ac i e agen o clinical use.19
To de e mine he esidual amoun s o Plu onic F68 on
PLGA NPs, 1H-NMR (500 MHz) spec a we e eco ded
a 303 K in CDCl3, on a B üke A ance 500 spec ome e
(B üke AXS Inc, Madison, WI). Chemical shi s we e
gi en in ppm, using he esidual p o ona ed sol en signal
as e e ence.
Di e en ial scanning calo ime y (DSC)
The physical s a us o CB13 inside he d ug-loaded PLGA
NPs was in es iga ed by DSC. The mog ams o CB13,
Plu onic F68, PLGA 50:50, and NPs (6% and 20% w/w d ug/
polyme ) we e eco ded on a DSC (Se a am 131; Se a am
Ins umen a ion, Calui e, F ance). Samples (5 mg weighed
o a p ecision o 0.005 mg, Model CP 225D; Sa o ius AG,
Goe ingen, Ge many) we e placed in aluminum pans, and
he lids we e c imped using a Shimadzu c impe . The mal
beha io o he samples was in es iga ed a a scanning
a e o 10°C/minu e co e ing a empe a u e ange o
25°C–300°C.
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In e na ional Jou nal o Nanomedicine 2012:7
S abili y s udy o NPs
I is well known ha NPs made o hyd oly ic deg adable
polyme s will deg ade o e ime. The e ec s o pH and
empe a u e ha e a c ucial e ec on long- e m s abili y.20 In
o de o e alua e he s abili y o PLGA NPs, a b ie s udy was
ca ied ou . Samples o blank and loaded NPs we e incuba ed
in Milli-Q wa e , phospha e-bu e ed saline (PBS) (pH 7.4),
and Dulbecco’s modi ied Eagle medium (DMEM) (Sigma-
Ald ich), a 4°C and 37°C. Samples we e collec ed a e
p e ixed incuba ion pe iods and cen i uged o 5 minu es
a 4000 pm in o de o p ecipi a e he agg ega es. Finally,
he mean pa icle size o he emaining nonagg ega ed NPs
was de e mined by lase sca e ing.21
F eeze-d ying p ocess
F eeze-d ying is a widely used p ocess o d ying and
imp o ing he s abili y o a ious pha maceu ical agen s.
As his p ocess is ela i ely slow and expensi e, i is usually
only used o p oduc s wi h a high added alue, which is he
case o mos ypes o NPs con aining expensi e ac i es,
such as CB13. The esul ing eeze-d ied NPs should ha e
ce ain desi able cha ac e is ics: he p ese a ion o he ini ial
cha ac e is ics o he p oduc , sho econs i u ion ime and
an accep able suspension, low o unmodi ied pa icle size
dis ibu ion o he NPs suspensions, and unchanged ac i i y
o encapsula ed d ug.
The e a e di e en ac o s han can a ec he inal quali y
o he p oduc : o mula ion, use o c yop o ec an , and he
s o age condi ions. F eezing is he i s s ep o eeze-d ying
and gene a es many s esses. Du ing his s ep, he liquid
suspension is cooled, and ice c ys als o pu e wa e o m –
he c ys alliza ion o ice may exe cise a mechanical s ess
on NPs, leading o hei des abiliza ion.
In he p esen wo k, he use o wo commonly employed
c yop o ec an s a di e en concen a ions was e alua ed:
p opylene glycol, a low molecula weigh alcohol; and
manni ol, a suga . Bo h we e added di ec ly o he NPs
suspension be o e eezing wi h liquid ni ogen. A e
his, NPs we e de os ed a RT. To e alua e he e ec o
each c yop o ec an , NP diame e s we e measu ed by lase
sca e ing.
De e mina ion o d ug inco po a ion
CB13 con en o he NPs was assessed di ec ly om he
ex ac ion o he d ug om NPs. The lyophilized NPs (abou
5 mg) we e accu a ely weighed using a high-p ecision
analy ical balance (d = 0.01 mg; Model CP 225D; Sa o ius).
Then, 1 mL ace oni ile was added, and he mix u e was
accu a ely o exed o dissol e he pa icles in he o ganic
phase. A e his, 10 µL o he solu ion was il e ed (Millex®
GV sy inge il e , 0.22 µm; Me ck Millipo e) and injec ed
in o he HPLC sys em o CB13 de ec ion.
The d ug con en was exp essed as EE (%) and d ug
loading (%) ollowing Equa ions 1 and 2:
EE Ac ualamoun o CB loaded in NPs
amoun o CB in
%=13
13Theo e ical NNPs




×100
(1)
D ug loadingwwMasso CB in NPs
Masso NPs ee ed
(% /)=



×
13
co 1100
(2)
In i o d ug elease
NP samples we e suspended in phospha e bu e (pH 7.4),
main ained a 37°C, and s i ed mechanically (100 pm)
du ing he elease expe imen s (Uni onic Vai én; Selec a
S.A., Ba celona, Spain). Aliquo s (500 µL) we e wi hd awn
a ixed ime in e als and il e ed upon cen i uga ion a
8000 pm. The il e ed sample (Millex GV) (10 µL) was
injec ed in o he HPLC appa a us o he e alua ion o
CB13.
Cell cul u e and in i o cy o oxici y
Cy o oxici y assays we e ealized using human no mal colonic
CCD-18Co cell lines and he human ca cinoma T-84 cell lines
ob ained om Ame ican Type Cul u e Collec ion (ATCC,
Manassas, VA). CCD-18Co cells we e g own in Eagle’s
minimal essen ial medium (MEM) (Sigma-Ald ich) wi h
Ea le’s balanced sal solu ion (BSS) and supplemen ed wi h
10% e al bo ine se um (FBS), 2 mM L-glu amine, 1 mM
sodium py u a e, and 14 mM NaHCO3. T-84 cells we e
g own in DMEM, supplemen ed wi h 10% FBS, 2 mM
L-glu amine, 15 mM HEPES, 14 mM NaHCO3. Gen amicin
(40 µg/mL) and ampicillin (500 µg/mL) (An ibió icos SA,
Mad id, Spain) we e used in bo h mediums. Cells we e
main ained in monolaye cul u e a 37°C in an a mosphe e
con aining 5% CO2. The cy o oxici y o CB13 loaded-PLGA
NPs (Resome 502) was e alua ed using a p oli e a ion assay;
a s udy o blank NPs and ee CB13 oxici y was included.
Cells we e seeded in a 24-well pla e (25 × 103 cells pe
well) and ea ed wi h CB13 loaded-PLGA NPs, blank NPs,
and ee CB13 a a wide ange o concen a ions. A 24 and
48 hou s, cells we e ixed wi h 10% ichlo oace ic acid o
60 minu es a 4°C and s ained wi h 0.4% sul o hodamine
B/1% ace ic acid by incuba ing o 10 minu es wi h
cons an shaking. Cells p e iously washed wi h 0.1%
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ace ic acid we e le in 10 mM T izma® (T6066-100G;
Sigma-Ald ich) o 15 minu es a oom empe a u e wi h
cons an shaking. The op ical densi y (OD) a 492 nm
was de e mined using a Ti e ek Mul iscan MC (Flow
Labo a o ies L d., I ine, Ay shi e, Sco land). To de e mine he
pe cen age o ela i e cell iabili y (%RCV) we used he o mula
%RCV = ( ea ed cells OD/un ea ed cells OD) × 100.
Resul s and Discussion
Cha ac e iza ion o he NPs
The size and size dis ibu ion o he NPs was measu ed by
lase sca e ing and he esul s a e epo ed in Table 1. The
da a a e exp essed as he mean diame e alue and s anda d
de ia ion (SD) ob ained om independen expe imen s.
The analysis showed ha he samples had a pa icle size
90–300 nm in diame e ega dless o he ini ial condi ions in he
p epa a ion, wi h a coe icien o a ia ion (CV) abou 30%. The
e ec o polyme ype, su ac an concen a ion, and ini ial d ug
amoun we e s udied. Resul s ob ained showed ha he main
in luence on pa icle size was he ype o polyme employed
du ing he pa icle p oduc ion. I can be seen ha o Resome
502 and 502H, pa icles we e 300 nm in diame e , and o 504,
504H, and 752S, pa icles we e a ound 100 nm in diame e .
The pa icle size o NPs p epa ed wi h Resome s
dec eased wi h inc ease in molecula weigh om 12,000 o
48,000 Da (502 s 504). These esul s can be explained by he
hyd ophobici y o he polyme employed. A high molecula
weigh , highe hyd ophobici y and smalle pa icle size we e
ob ained due o longe alipha ic chains.22,23 No di e ences
in size we e ound due o he p esence o e minal ca boxyl
g oups in bo h cases.22 Compa ing esul s ob ained o R502
and R752S (simila molecula weigh ), smalle diame e s
we e ob ained when R752S was used. In his case, he lac ic
acid po ion inc eased up o 75%.
The ep oducibili y o he p ocedu e was also e alua ed.
In e lo CV% was in all cases less han 10%, which poin s
o a eliable p ocedu e o NP p oduc ion.
The ZP alues o he NPs shown in Table 1 indica e he
nega i e cha ges on he NP ba e su ace, which is due o
he o e all nega i e cha ges o unc ional g oups on PLGA
in Milli-Q wa e . The high nega i e su ace change is an
impo an indica ion o he s abili y o a colloidal sys em in
medium. The epulsion among he highly nega i ely cha ged
NPs p o ides ex a s abili y. The ZP o ou o he ypes o
NPs was measu ed below –24 mV, p o ing he high s abili y
o he NPs suspended in aqueous medium. When CB13-
PLGA NPs we e e alua ed, a lowe ZP alue was ob ained
(–20 mV; da a no shown).
The use (o no ) o a su ac an on he o ganic phase also
s ongly a ec ed he pa icle size o NPs elabo a ed wi h
Resome 502 and 502H (see Table 2). I can be seen ha
when no su ac an was employed o i s concen a ion was
oo low, pa icle diame e inc eased up o 600 nm, and he e
was a highe deg ee o polydispe si y.
The ini ial amoun o CB13 was also e alua ed. No in lu-
ence on pa icle size o pa icle size dis ibu ion was ound
(see Table 3).
As an example, Figu e 1 shows a SEM mic opho og aph
o he PLGA NPs. The pic u e e ealed a egula and sym-
me ical mo phology, and a e p ocessing he SEM image,
he size o he esul ing pa icles was be ween 170 and
230 nm (by lase sca e ing analysis).
D ug loading
The capaci y o d ug loading o he NPs is an impo an
ac o in hei o mula ion since high loading implies smalle
amoun s o he NPs a e needed o a gi en dose o he
ea men . The CB13 loading (%w/w) as well as he EE%
a e epo ed in Table 4.
In e ms o EE%, high alues we e achie ed o all
NPs p epa ed due o he poo solubili y o CB13 in he
ex e nal aqueous phase,24 and highes alues we e achie ed
using Resome 502. Panyam e al25 demons a ed ha when
hyd ophobic d ugs a e used, NPs d ug loading is closely
Table 1 Pa icle mean diame e , size dis ibu ion, and ZP alues
ob ained as a unc ion o polyme used (n = 6)
Polyme Dmean
(nm)
SD
(nm)
CV (%)
in alo
CV (%)
in e lo
ZP ± SD
(mV)
R502 311.02 12.94 34.2782 4.16 −24.5 ± 2.3
R502-H 281.37 11.30 34.628 4.02 −42.6 ± 0.6
R504 89.33 0.96 35.0231 1.08 −28.4 ± 1.1
R504-H 91.97 10.38 34.8708 2.29 −44.8 ± 0.9
R752S 131.06 6.02 38.1749 4.60 −33.2 ± 0.7
Abb e ia ions: R, Resome ®; Dmean, mean diame e ; SD, s anda d de ia ion;
CV (%), coe icien o a ia ion pe cen age; ZP, ze a po en ial.
Table 2 Pa icle mean diame e and size dis ibu ion o he nano-
pa icles as a unc ion o su ac an concen a ion assayed (n = 3)
Polyme Csu (%w/ ) Dmean (nm) SD (nm) CV (%)
R502 0 592.60 296.10 49.97
0.1 462.26 533.70 115.45
0.5 304.22 111.50 36.65
R502-H 0 600.56 219.89 36.61
0.1 269.40 96.70 35.89
0.5 287.35 116.38 52.22
Abb e ia ions: R, Resome ; Csu , su ac an concen a ion; Dmean, mean diame e ;
SD, s anda d de ia ion; CV (%), coe icien o a ia ion pe cen age.
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Table 3 Size and size dis ibu ion o he nanopa icles as a
unc ion o ini ial amoun o CB13 employed (polyme used:
Resome 502) (n = 3)
CB13 (%w/ ) Dmean (nm) SD (nm) CV (%)
0 291.28 99.8 34.2782
10 321.73 108.9 33.857
20 315.25 108.3 34.360
Abb e ia ions: CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone;
Dmean, mean diame e ; SD, s anda d de ia ion; CV%, coe icien o a ia ion
pe cen age.
18
15
10
5
0
0.010 0.100 1.000 10.00
Diame e (µm)
q (%)
100.0 1000 3000
AB
Figu e 1 (A) Typical size dis ibu ion o CB13-PLGA NPs (polyme used: Resome 502, loading: 10% w/w). (B) SEM pho og aph o he same o mula ion.
Abb e ia ions: CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone; PLGA, poly(lac ic-co-glycolic acid); NPs, nanopa icles; SEM, scanning elec on mic oscope.
ma ched wi h he espec i e solid-s a e d ug–polyme
solubili y, ha is, he abili y o he polyme ic ma ix o en ap
d ug in he dispe sed s a e. The solid-s a e solubili y o he
d ug in he polyme inc eased wi h an inc ease in he lac ide
con en in he polyme and wi h a dec ease in he molecula
weigh . Mo eo e , he p esence o ee acid end g oups in
he polyme esul ed in a dec ease in he d ug’s solubili y in he
polyme . This can be explained on he basis o he inc easing
hyd ophobici y o he polyme wi h inc easing lac ide con en
o wi h es e end g oups, esul ing in be e solid-s a e solubili y
o he hyd ophobic d ug in he hyd ophobic polyme ma ix.
So, in ou s udy his hypo hesis is ue. NPs elabo a ed
wi h Resome 502 showed he highes alues in d ug load-
ing due o hei lowe molecula weigh and he p esence o
he es e i ied end g oups. On he o he hand, NPs elabo a ed
wi h Resome 504H showed he lowes alues due o hei
highe molecula weigh and he p esence o ee acid end
g oups. Finally, NPs elabo a ed wi h Resome 752S showed
simila d ug loading alues o hose ob ained using Resome
502, whe e he e ec s o molecula weigh and lac ide con en
would be balanced.
In ela ion o he ini ial amoun o CB13 employed
(6, 10, o 20%w/w), EE dec eases when CB13 ini ial con-
cen a ion inc eases. This means ha he e is a maximum
quan i y o d ug ha can be en apped in he polyme
ma ix in he dispe sed s a e; ha is, he e is a limi o d ug
miscibili y in he polyme .26 The emainde o he d ug can
mig a e o he su ounding emulsi ie aqueous phase. As
was indica ed by Panyam e al,25 i he su ac an u ilized o
s abilize he emulsion is p esen a a concen a ion g ea e
han i s c i ical micella concen a ion, su ac an micelles
can solubilize he d ug, esul ing in a loss o encapsula ion
by he NPs. In ou case, he concen a ion o su ac an -
used, Plu onic F68 (0.06 mM), was sligh ly abo e i s c i ical
micella concen a ion (0.04 mM), which may explain his
phenomenon.
In ela ion o he CB13 loading (%w/w), his seems o
inc ease wi h he ini ial amoun o d ug employed du ing
he p epa a ion o he NPs. Se e al s udies showed ha d ug
loading can be enhanced by inc eased ini ial amoun o d ug.
Howe e , EE does no necessa ily inc ease wi h highe ini ial
amoun o d ug. Once he maximum loading capaci y o NPs
is eached, u he inc ease in ini ial amoun o d ug can e en
dec ease he EE.27
Di e en ial scanning calo ime y (DSC)
The physical s a es o he d ug in he NPs ha could in luence
he in i o and in i o elease o he d ug om he sys em
was in es iga ed by DSC.28 Figu e 2 shows he DSC he -
mog ams o pu e CB13 powde , CB13-loaded PLGA NPs,
and blank PLGA NPs. Plu onic F68 and PPG he mog ams
we e also analyzed.
The mel ing endo he mic peak o pu e CB13 appea ed
a 69.55°C (∆H = 64,651 J/g). Howe e , no mel ing peak
was de ec ed o bo h NP o mula ions. Resome 502 and
Plu onic F68 p esen ed endo he mic peaks a 45.90°C and
53.07°C, espec i ely. A Plu onic F68 peak was no de ec ed
o NPs. Thus, i can be concluded ha CB13 inside he NPs
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Table 4 D ug loading and encapsula ion e iciency o NPs (n = 6)
Polyme CB13
(%w/w)ini ial
EE ± SD (%) CB13 loading ± SD
(%w/w)
R502 6 89.61 ± 0.25 6.16 ± 1.12
10 82.26 ± 3.73 12.22 ± 6.69
20 70.75 ± 4.82 16.26 ± 0.97
R502H 6 80.74 ± 2.95 4.84 ± 1.12
10 77.094 ± 3.01 6.71 ± 2.30
20 67.709 ± 1.98 15.64 ± 3.01
R504 6 75.44 ± 2.79 16.90 ± 1.10
R504H 6 71.53 ± 3.06 16.11 ± 2.24
R752S 6 83.21 ± 1.57 18.75 ± 3.05
Abb e ia ions: NPs, nanopa icles; R, Resome ; CB13, 1-Naph halenyl[4-(pen yloxy)-
1-naph halenyl]me hanone; EE, encapsula ion e iciency; SD, s anda d de ia ion.
−30
−25
−20
−15
−10
−5
0
5
10
15
20
25
30
29.3
29.9
38.4
50.3
61.9
73
83.8
94.4
105
116
126
137
147
158
168
179
189
200
210
221
231
242
252
263
273
284
294
305
315
T (ºC)
Hea low
Resome 502 F ee CB13 Loaded NPs Plu onic F68 PPG Blank NPs
Figu e 2 DSC cu es o Resome 502, ee CB13, loaded-PLGA NPs (20% w/w), Plu onic® F68, PPG, and blank-PLGA NPs.
Abb e ia ions: DSC, di e en ial scanning calo ime y; CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone; PLGA, poly(lac ic-co-glycolic acid); NPs, nanopa icles;
PPG, p opylene glycol.
was ei he in an amo phous, diso de ed c ys alline phase o
in he solid solu e.19,29
Residual amoun o Plu onic F68
The e a e se e al me hods o de ec esidual amoun s o su ac-
an s. One o he mos popula is a colo ime ic me hod de el-
oped by Childs,30 employed also o quan i y esidual amoun s
o Plu onic F68. The colo ime ic me hod is based on he o -
ma ion o a colo ed complex be ween wo hyd oxyl g oups o
Plu onic F68, Ba2+, and an iodine molecule. Then, he complex
is e alua ed by measu ing i spec opho ome ically a 540 nm.
In he p esen wo k, he de e mina ion o he esidual
amoun s o Plu onic F68 was ca ied ou by 1H NMR spec-
oscopy.31 The 1H NMR spec um o CB13, Plu onicF68,
Resome 502, and blank and loaded PLGA NPs a e shown
in Figu es 3 and 4.
The cleaned NPs we e collec ed by cen i uga ion and
lyophilized. The powde ob ained was dissol ed in CDCl3
o 1H NMR analysis. The esidual Plu onic F68 adso bed
on o pa icles was expe imen ally es ima ed acco ding o he
ollowing equa ion:31
Nmgg M
M
I
I
PF
PLGA
PF
PLGA
(/)(.)
(..)
=



×
=
=−
68 68 117
535515
87
δ
δ
11000,
(3)
in which IPLGA was he peak in eg al o he
–
CH g oups o
he lac ide uni om Resome 502 (δ = 5.35–5.15 ppm)
co esponding o 1 p o on; IPF68 was he peak in eg al o
he
–
CH3 g oups o polye hylene oxide uni om Plu onic
F68 (δ = 1.17 ppm) co esponding o 87 p o ons; MPLGA was
he sum o he molecula weigh s o he lac ide and he gly-
colic uni s in PLGA (130 g/mol); and MPF68 was he molecula
weigh o he Plu onic F68 (8350 g/mol).
Addi ionally, 1H-NMR spec a we e analyzed o quan i y
he amoun s o CB13 p esen in NPs, ollowing Equa ion 4.
Resul s we e exp essed as Q (w/w%):
QM
M
I
I
CB
PLGA
CB
PLGA
(%) /
()
()
=



×
=
=
13 13 12
52
3100
δ
δ
.
.
(4)
whe e MCB13 was he molecula weigh o CB13 (368.47 g/mol)
and MPLGA was he sum o he molecula weigh s o he lac ide
and he glycolic uni s in PLGA (130 g/mol). ICB13 was he peak
in eg al o he
–
CH3 g oups (δ = 1.2 ppm) co esponding o
3 p o ons and IPLA was he peak in eg al o he
–
CH g oups
o he lac ide uni (PLA [polylac ic acid]) om Resome 502
(δ = 5.35–5.15 ppm) co esponding o 1 p o on.
These esul s acco ded wi h HPLC esul s o CB13
loading in Resome 502 NPs (see Table 4).
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In e na ional Jou nal o Nanomedicine 2012:7
10 987654321 ppm
3.01
2.03
6.13
1.99
2.03
1.00
3.03
3.01
1.03
1.00
1.01
1.00
1.01
1.00
A
B
8.07.5 7.06.5 6.05.5 5.04.5 4.03.5 3.02.5 2.01.5 1.00.5 0.0ppm
3.81
1.92
1.00
Lac i
O
O
O
O
xy
O
**
CH3
O
Glico
Figu e 3 (Con inued)
F eeze-d ying
NP in eg i y a e he eezing p ocess in liquid ni ogen
was s udied unde di e en expe imen al condi ions in o de
o de e mine he op imal concen a ion o c yop o ec an .
C yop o ec an s a e ypically small, highly hyd oxyla ed
molecules, such as monosaccha ides o low molecula
weigh alcohols, ha can be added o he suspension p io
o lyophiliza ion o p ese e he mo phological in eg i y o a
nanoma e ial du ing eeze-d ying. We he e o e wen on o
explo e an op imal concen a ion o wo o hese subs ances,
p opylene glycol and manni ol. The amoun s o he c yop o-
ec i e agen we e a ied om 0 o 50 w/w%, by dissol ing
he agen di ec ly in o he NP suspension immedia ely p io
o eeze-d ying.
As can be seen in Figu e 5, he suspension wi hou c yo-
p o ec an o med s icky gels du ing eeze d ying. These
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C
9.08.5 8.07.5 7.06.5 6.05.5 5.04.5 4.03.5 3.02.5 2.01.5 1.00.5 ppm
3.00
1.03
23.47
0.20
0.11
—CH2
HOOOOH
CH3
aab
Figu e 3 1H NMR spec um o he CB13 (A), Resome 502 (B), and Plu onic F68 (C).
Abb e ia ion: CB13, 1-Naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone.
lyophilized gels could no be esuspended in Milli-Q wa e ,
PBS, no DMEM unde any o he condi ions ied (including
ul asonic agi a ion).
The pa icle size a e he eezing p ocess was e alu-
a ed measu ing mean pa icle diame e by lase sca e ing.
Figu e 6 shows he e ec o he addi ion o c yop o ec an
on NPs size and size dis ibu ion.
Resul s ob ained o manni ol, sugges ed ha he op imal
concen a ion was no eached and ha NP diame e s we e
dec easing wi h inc easing manni ol concen a ion. When
p opylene glycol was used, he amoun o 100 µL o p o-
pylene glycol was ound o be op imal (NPs diame e and
CV% we e close o ini ial pa icle diame e ).
NP s abili y
Fo a be e unde s anding o he in i o elease p o iles, NP
s abili y in di e en incuba ion media a 37°C was analyzed.
Con e sely, o de e mine he op imal s o age condi ions,
s abili y s udies a 4°C we e also ca ied ou . Figu e 7 shows
he NP size dis ibu ion a di e en imes in Milli-Q wa e ,
PBS, and DMEM a 4°C o 37°C.
As e e ence, he g een (and ed) line indica es he ini ial
pa icle size (300 ± 102 nm; = 0 hou s). A e incuba ion
in PBS a 37°C o 6 hou s (black line), NP size inc eased
o up o 100 µm in diame e . In PBS a 4°C o 6 hou s
(blue line), a bimodal pa icle size dis ibu ion was ob ained.
A small ac ion o NPs we e up o 100 µm in diame e ,
bu he majo % was a ound 700 nm in diame e . These
esul s poin ou a slowe deg ada ion o NPs in PBS a 4°C
s 37°C. Ne e heless, compa ing his esul wi h s abili y
in Milli-Q wa e a 4°C, he esul is exac ly he con a y.
A bimodal pa icle size dis ibu ion was ob ained, wi h a
small ac ion o NPs a a ound 1 µm. In DMEM a 37°C
incuba ion, esul s we e simila o hose ob ained in PBS a
37°C (da a no shown).
In i o elease
The wo main elease mechanisms associa ed wi h d ug
elease om PLGA-based deli e y sys ems a e di usion
and deg ada ion/e osion. The elease a e is o en said o be
di usion con olled ini ially and deg ada ion/e osion con-
olled du ing he inal s age o he elease pe iod. Howe e ,
many ac o s in luence he a e o d ug di usion and he
deg ada ion kine ics, o example, polyme –d ug in e ac-
ions, d ug–d ug in e ac ions, wa e abso p ion, and po e
closu e. An ex ensi e e iew ocusing on he mechanisms o
d ug elease om PLGA-based deli e y sys ems has been
ealized by F edenbe g e al.32
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